Mouse vendor-dependent variation in gut microbiota determines susceptibility to Salmonella infection Eric M. Velazquez and Andreas J. Bäumler Medical Microbiology and Immunology, University of California, Davis, CA

Abstract ResultsResults The gut microbiota contributes to intestinal health and can protect its host against Figure 1 Figure 3 diarrheal infections. However, specific members of the microbial community that confer protective benefits are not fully described. We hypothesized that specific 11 10 A. PC2 B. 100 microbiota differences between healthy individuals can be associated with more Verrucomicrobia 9 90 resistance to enteric pathogens. We first tested if genetically similar strains of mice 8 Proteobacteria Salmonella % 80 (log CFU 7 Abundance obtained from different commercial vendors exhibit different responses during Taconic Tenericutes 6 of fecal 70 Salmonella infection. C57BL/6 mice from Harlan and Jackson were orally challenged per gram C. River feces) 5 community 60 Firmicutes with increasing doses of Salmonella. At each given dose, pathogen loads were 4 analyzed by 50 Deferribacteres 3 16S rRNA consistently higher in Jackson mice compared to Harlan mice. Next, we directly Harlan 40 2 sequencing Cyanobacteria tested if the difference in Salmonella colonization was microbiota-dependent. 1 Jackson 30 Bacteroidetes Colonizing germ-free mice with fecal transplants recapitulated the infective dose 0 20 % mice infected: 0 0 100 0 75 100 0 100 100 100 100 100 10 Actinobacteria response associated with each donor. Microbiome sequence analysis identified PC1 0 Proteobacteria in donor and recipient animals from Harlan, but not Jackson. We 5 7 9 5 7 9 5 7 9 5 7 9 Challenge dose: 10 10 10 10 10 10 10 10 10 10 10 10 Har CR Tac Jax investigated the causal role of these commensal bacteria by transferring them into PC3 Mouse vendors: Harlan C. River Taconic Jackson Jackson mice. After being colonized with Enterobacteriaceae isolates, Jackson mice Phylum-level Differences Correlate with Host Susceptibility showed improved intestinal resistance against Salmonella. Importantly, these Specific Pathogen-Free C57BL/6 Mice Obtained from Different Vendors 16S rRNA sequencing of feces from uninfected mice reveals that relatively resistant findings suggest that the natural levels of Enterobacteriaceae among healthy groups cluster away from the more susceptible ones (3A: Unweighted PCA plot). Intestinal Salmonella Colonization Varies by Mouse Supplier individuals may determine susceptibility during a foodborne outbreak. Jackson mice in particular lack detectable Proteobacteria. (3B: Abundance columns Animals were allowed to acclimate in our vivarium for at least one month before any of all mice from each given vendor were averaged). Data were analyzed in QIIME. Background experiments were started. Infections were performed by oral gavage with increasing doses of Salmonella. Fecal pellets were collected at 4 days post-infection and Figure 4 • During a foodborne outbreak, people Cases per million pathogen loads were enumerated by plating. Mice from Jackson have the lowest population can exhibit a range of illness, from implantation dose needed to become infected. They also have greater burdens of Commensal Enterobacteriaceae Salmonella spp. 0.1 – 1.9 self-limiting gastroenteritis to death. Salmonella at each given inoculum. Genetic or microbial differences between 2 – 4.9 7 7 5 – 9.9 mouse substrains could be contributing to the variable susceptibility observed. 6 6 • Attack rates may be variable due to 10 – 19.9 5 5 specific host, pathogen, and >20 Bacteria 4 4 (CDC, 2008 Outbreak) Figure 2 (log CFU environmental interactions. 3 3 per gram Pathogen virulence 2 2 • We hypothesize that natural 11 feces) Dose ingested 10 1 1 n.d. n.d. n.d. n.d. variation in the gut microbiota 9 0 0 Host genetics between healthy individuals Salmonella 8 CR Har Tac Jax CR Har Tac Jax may underlie differences in Immune system (log CFU 7 Jackson mice given MacConkey isolates from: susceptibility during intestinal Diet and stress per gram 6 feces) 5 Gut microbiota Salmonellosis Salmonella infection. 4 Indigenous Proteobacteria Exclude Salmonella Implantation 3 Fecal transplants of uninfected mice from different vendors were grown aerobically Methods 2 on MacConkey agar and colonies with unique morphologies were isolated. Mice 1

0 from Jackson that were colonization with these cultured strains and subsequently Experimental Model % mice infected: 0 33 100 33 25 100 0 100 100 75 100 100 challenged with a 105 inoculum of Salmonella were protected. (n.d., not detectable)

Donors with Most resistant Challenge dose: 105 107 109 105 107 109 105 107 109 105 107 109 different microbiota to infection Summary Mouse vendors: Harlan C. River Taconic Jackson Infect Figure 1 Figure 3 S R R • Mice from different vendors have varying • The intestinal niche for Germ-Free Swiss Webster Mice Given Feces from Different Vendors degrees of colonization resistance and this Enterobacteriaceae is occupied Plate Sequence property is transmissible via the microbiota. on a first come, first served basis. Infectious Dose Response is Determined by Gut Microbes Feces from mice used in Figure 1 (before infection) were given to inbred germ-free Fecal transfer Isolate bacteria mice. These recipients animals were maintained under sterile conditions for the Acknowledgements entire experiment. After allowing 5 days for the fecal transplant to establish, Keaton Heasley (CSU Northridge) for data analysis and graphics. Infect Figure 2 Infect Figure 4 recipient mice were challenged with increasing doses of Salmonella. The percent of Christopher Lopez and Franziska Faber for animal husbandry. S R S animals infected and the colonization levels of Salmonella at each tested inoculum Veterinary Scientist Training Program for DVM support. Plate Plate were consistent between the donors in Figure 1 and the respective recipients. Integrative Pathobiology Graduate Group for PhD support. These findings support the idea that differences in the baseline level of colonization Germ-free recipients Most susceptible Animal Models of Infectious Diseases Training Program for funding. with same genetics to infection resistance between mice from various breeders is microbiota-dependent. Students Training in Advanced Research (STAR) Summer Program for funding.